Vibrio cholerae is the causative agent of the dehydrating diarrheal disease, cholera. For disease to occur, virulence genes including those encoding cholera toxin and toxin co-regulated pilus must be appropriately expressed. Expression of virulence genes is coordinately regulated by a complex cascade of transcriptional activators in response to environmental signals. ToxR and TcpP, two membrane-localized transcription factors, are required for activation of toxT transcription, and ToxT directly activates expression of virulence genes. The goals of this application are to further investigate the structure-function and localization requirements of the transcriptional activators, ToxR and TcpP. Specifically, we aim to determine the cellular localization of these transcriptional activators and the promoter DMA they specifically bind using cytological techniques. We will also identify the mechanism controlling TcpP turnover in the absence of TcpH through the development of a genetic screen and analysis of periplasmic domain interactions. In addition, studies using the infant mouse model will characterize the in vivo effects of altering TcpP turnover to test the hypothesis that regulating protein levels is important for V. cholerae colonization. [unreadable] [unreadable]